To 10 ug DNA linearized and dissolved in 40 ul H2O, add 10 ul 5 X CB CIP and
1 unit of calf intestinal phosphatase (CIP). You need .01 units
CIP to dephosphorylate 1 pmole of 5' ends (1 pmole of 5' ends of a 4 kb DNA fragment
is 1.6 ug). Thus, I use a slight excess of CIP.
Unlike BAP, incubations with CIP are done at 37°C for 5' overhangs for 1/2 hr
with two additions, or at 37°C for 1/2 hr then at 56°C for 1/2 hr after another addition, for 3' overhangs or blunt DNA. In all cases the reaction is terminated by the addition of 2.5
ul 20% SDS, 40 ul H2O, 10 ul 10 X STE (TNE) vortexed and heated at 65°C for 15
mins followed by multiple phenol chloroform extractions and ethanol precipitation.
Always do control transformations of vector before phosphatase
religated, vector after phosphatase religated and not religated to determine the
efficiency of linearization in the first place, as well as the efficiency of
dephosphorylation. The dephosphorylated vector should not be able to religate to itself
and thus needs a phosphorylated fragment to contribute the 5' phosphate at each end
to create a nicked but circular molecule (catalyzed by DNA ligase) that can transform
with moderate efficiency.
If you get no colonies with addition of insert to ligation reaction with
phosphatased vector, you need to worry about the possibility that a nucleolytic
contaminant of the phosphatase has destroyed your sticky ends, or that residual
phosphatase activity is dephoshorylating your insert in the ligation reaction, versus a
problem with the insert, e.g. inhibition of ligation due to some poison from agarose,
acrylamide gels or centrifuge desalting or elution columns, etc. The control in this case
is to ligate your phosphatased vector to the digest of a plasmid with a different
antibiotic resistance marker which also contains a reasonable sized (100 to 1000 bp)
similarly sticky ended insert. Thus, without purifying the DNA you can mix the digest
with the phosphatased vector, ligate, select for the phosphatased vectors antibiotic
resistance and see if sticky fragments were able to "rescue" the phosphatased vector.
If yes, then the problem is with your insert, if no, then the problem is with your